Endothelium-derived relaxing factor (EDRF), is a product of the normal endothelial cell, and has both vasodilatory and antiplatelet properties (Furchgott, R. F. et al., Nature, 288:373-376 (1980); Moncada, S. et al., Biochem. Pharmacol, 38:1709-1713 (1989); Azuma, H. et al., Brit. J. Pharmacol. 88:411-415 (1986) and Radomski, M. W. et al., Brit. J. Pharmacol. 92:639-642 (1987)). Pharmacologic studies suggest that disease states as varied as septic shock, hyper-homocysteinemia, atherosclerosis, and hypoxia-induced pulmonary hypertension may be associated with abnormal concentrations of EDRF in the vascular milieu (Westernberger, U. et al., Free Rad. Res. Comm. 11:167-168 (1990); Yamamoto, H. et al., J. Clin. Invest. 81:1752-1758 (1988); Dinh-Xuan, A. T. et al., Engl. J. Med. 324:1539-1547 (1991)). This bioactive substance is believed to be equivalent to nitric oxide, or a chemical congener or adduct thereof (Palmer, R. M. G. et al., Nature 327:524-525 (1987); Ignarro, L. J. et al., Proc. Natl. Acad. Sci. 84:9265-9269 (1987)). Among the species of importance as biological adducts of nitric oxide are S-nitrosothiols, which are adducts with the sulfhydryl groups of amino acids, peptides, and proteins.
It has been demonstrated that nitric oxide and authentic EDRF react with free thiol groups of proteins under physiologic conditions in vitro, to form S-nitroso-proteins. These nitric oxide adducts have bioactivities which are comparable to nitric oxide, but exhibit half-lives on the order of hours, significantly longer than that of EDRF (Stamler, J. S. et al., Proc. Natl. Acad. Sci. 89:444-448 (1992)).
Under normal circumstances, the concentration of nitric oxide in blood or plasma is believed to be quite low (in the 1 nM range) and its half-life of the order of 0.1 second. Its high degree of reactivity toward oxygen and redox metals, in conjunction with its extremely short half-life, have made the routine measurement of blood levels in both normal and disease states most difficult by standard methods, such as chemiluminescence spectroscopy, electron paramagnetic resonance spectroscopy, or differential absorbance spectroscopy of hemoglobin (Martin, W. et al., J. Pharmacol. Exp. Therap. 237:529-538 (1986); Downes, M. J. et al., Analyst 101:742-748 (1976); Kelm, M. et al., Circ. Res. 66:1561-1575 (1990); Arroyo, C. M. et al., Free Rad. Res. Comm. 14:145-155 (1991) and Goretsky, J. et al., J. Biol. Chem. 263:2316-2323 (1988)). In fact, it is generally assumed in the field that such measurements are not feasible by currently used methods.
Nitrosonium (NO.sup.+) is a short lived species which is too unstable to exist freely in biological systems, and felt to be non-detectable by chemiluminescence. Nitric oxide exists in the S-nitrosothiol adduct, not as nitric oxide but rather as a nitrosonium equivalent. Thus, it behaves chemically in a manner which more closely resembles NO.sub.+ than NO.sup.- (nitric oxide).
Hemoglobin (Hb) is a tetramer comprised of two alpha and two beta subunits. In human Hb, each subunit contains one heme, while the beta (.beta.) subunits also contain highly reactive thiol (SH) groups (cys.beta.93) (Olson, J. S., Meth. of Enzym., 76:631-651 (1981; Antonini & Brunori, In Hemoglobin and Myoglobin in Their Reactions with Ligands, American Elsevier Publishing Co., Inc., New York, pp.29-31 (1971)). These cysteine residues are highly conserved among species. Nitric oxide (NO) interacts with hemoglobin at its metal centers, whereas S-nitrosothiols (RSNOs) can donate the NO group to .beta.93 cysteine residues.
Nitric oxide is known to bind tightly to hemoglobin, forming nitrosyl(FeII)-hemoglobin. Interactions of NO with ahemoglobin are believed to be a major route of NO metabolism in biological systems. It follows that levels of NO-hemoglobin in blood should be an excellent indication of endogenous NO production. However, methods have not been developed that are sufficiently sensitive to make this determination in vivo (Beckman, J. S. et al., Methods in Nitric Oxide Research, Feelisch and Stainler, J. S. eds, Wiley, Chichester, U.K. (1996)). Specifically, electron paramagnetic resonance (EPR) has been used previously to measure nitric oxide bound to the Fe of the heme. However, under normal physiological conditions, circulating levels of NO-hemoglobin in blood are below the detection limit. The insensitivity of EPR makes this method impossible to use to monitor all but a gross change in NO-hemoglobin from normal levels. Only in pathophysiological states such as sepsis and pregnancy, which are characterized by NO overproduction, can EPR be used to detect a measurable level for NO in blood.
EPR measurements also suffer from being cumbersome and expensive. An alternative method of measuring NO by assaying nitrite/nitrate in body fluids also suffers from insensitivity.